Process of producing highly transformable bacterial cells and cells produced thereby

ABSTRACT

The invention provided herein includes gram negative bacteria cells containing a gene encoding an enzyme with carbohydrate degrading activity that had been rendered competent to transformation. Carbohydrate degrading enzymes of interest for use in the invention include alpha-amylase. The competent cells of the subject invention may be frozen so as to provide for prolonged storage. Other aspects of the invention include methods for rendering gram negative bacterial cells, such as E. coli cells competent to transformation. These methods involve the step of transferring a gene encoding an enzyme with carbohydrate degrading activity into E. coli cells and subsequently rendering the cells competent using any of a variety of competency inducing procedures.

TECHNICAL FIELD OF THE INVENTION

The invention relates generally to the field of genetic engineering.More specifically, the invention relates to highly transformablebacterial cells and methods for producing such cells.

BACKGROUND OF THE INVENTION

A critical step in the in vitro formation of recombinant DNA constructsand the production of genetic libraries, as well as other geneticengineering techniques, is the process of inserting DNA (and othersimilar polynucleotides) into host cells. The process of introducingpolynucleotides into host cells is referred to as transformation.Bacterial cells are frequently used as host cells for a wide variety ofgenetic engineering experiments. Among the most frequently used speciesof bacteria for genetic engineering experiments is the organismEscherichia coli (E. coli). E. coli does not naturally import exogenousDNA into the cell. E. coli must be subjected to a process that rendersit amenable to uptake of exogenous DNA, i.e., a competency inducingprocedure. E. coli, as well as other bacterial cells, that have beenrendered capable of taking up exogenously added DNA are referred to ascompetent cells.

There are many established procedures for rendering E. coli cellscompetent. These procedures include the CaCl₂ incubation methods ofMandel and Higa, J. of Mol. Biol. 53:159 (1970), as well as numerouswell-known variants thereof. Hanahan has made a detailed study offactors that effect the efficiency of transformation of E. coli cells(J. Mol. Biol. 166:557-580 (1983)) where he describes a method ofproducing highly competent E. coli cells comprising the step of washingE. coli cells in a buffer comprising potassium acetate, KCl, MnCl₂,CaCl₂, and hexamine cobalt chloride, which is generally regarded as thebest available method of producing highly competent E. coli. Anothermethod of producing competent E. coli cells is described by Jessee etal., U.S. Pat. No. 4,981,797. Jessee et al. shows that high levels ofcompetency may be induced by growing E. coli cells in a temperaturerange of 18° C. to 32° C. as part of a competency inducing process.

The various techniques for rendering E. coli cells competent producecompositions of competent E. coli cells that vary widely intransformation efficiency. The mechanism by which DNA enters competentE. coli is not completely understood. Nor is it completely understoodwhy one composition of competent E. coli cells differs in transformationefficiency from that of another composition of competent E. coli cells.Hanahan, in Escherichia Coli and Salmonella Typhimurium: Cellular andMolecular Biology, editor F. C. Neidhardt, American Society forMicrobiology, Washington, D.C. (1987), provide a review of what is knownabout E. coli transformation. Only a fraction of the cells in apreparation of competent E. coli cells, i.e., a higher percentage ofcells capable of being transformed, are necessarily competent for DNAuptake. Thus, some superior methods of generating competent E. coli cellcompositions may simply result in the formation of competent cellcompositions that contain a higher percentage of competent E. coli cellsas opposed to containing E. coli cells that show individual levels ofhigher rates of DNA uptake or the ability to take up larger pieces ofDNA. Alternatively, other methods of producing competent cells mayresult in the formation of competent E. coli cells that "individually"have higher transformation efficiencies. Hanahan, in J. Mol. Bio.166:557-580 (1983) has speculated that competent E. coli cells containchannels for transport of DNA across the cell envelope and that thelimiting step in determining the competency for transformation of E.coli cells are events that occur in the cell after the cell has taken upthe DNA of interest, i.e., the establishment step. Another factoraffecting the transformation efficiency of a composition of competent E.coli cells is the genotype of the cells. Some strains of E. coli areknown to produce more highly transformable competent cell compositionsthan other strains of E. coli when subjected to the same competencyinducing procedure. Hanahan, U.S. Pat. No. 4,851,348, describes how E.coli deoR mutants can be used to produce highly transformable E. colicell compositions using a variety of competency inducing procedures.

Ever since it has been demonstrated that E. coli could be renderedcompetent to transformation, it has been of interest to produce the mostcompetent E. coli cell preparations possible. The maximum level oftransformation efficiency obtained using the method of Hanahan describedin J. Mol. Bio. 166:557-580 (1987), which employs the step of washingcells in a buffer comprising potassium acetate, KCl, MnCl₂, CaCl₂,glycerol, and hexamine cobalt chloride, is approximately 1×10⁹transformants per microgram of supercoiled pUC18 plasmid DNA. On a percell basis, this translates to approximately 1 cell out of 30 in thepopulation actually becoming transformed. However, there continues toexist a need for new and improved methods for producing competent E.coli of superior transformability, as well as new strains of E. colithat demonstrate superior transformability. Such methods and strainswould be of wide interest to most researchers in the field of geneticengineering in that the number of transformations required to obtain thedesired result would be minimized. Thus, for example, larger geneticlibraries could be built more easily as well as the construction ofcomplex recombinant molecules achieved more readily.

SUMMARY OF THE INVENTION

The invention described herein provides a method of producing highlytransformable gram negative bacterial cells such as E. coli that can beadapted to a wide variety of competency inducing procedures. The methodsof the subject invention involve the introduction of geneticconstructions containing a polynucleotide sequence encoding (and capableof expressing) an enzyme with carbohydrate degrading activity,preferably starch degrading activity. The enzyme is preferably locatedin the periplasmic space of the cell; however, the practice of theinvention is not dependent upon any particular theory as to the cellularlocation of expressed carbohydrate degrading enzymes. Compositions ofcompetent E. coli cells containing a genetic construction for theexpression of an enzyme with carbohydrate degrading activity are shownto be more readily transformed with exogenous DNA than similar E. colicells lacking such genetic constructions.

The invention provided herein includes E. coli cells containing apolynucleotide sequence encoding an enzyme with carbohydrate degradingactivity that has been rendered competent to transformation. Thecarbohydrate degrading enzymes of interest include alpha-amylase,particularly alpha-amylase isolated from a thermophilic bacterium. Thecompetent cells of the subject invention may be frozen so as to providefor prolonged storage.

Other aspects of the invention include methods for rendering gramnegative bacteria, such as E. coli cells competent to transformation.These methods involve the step of transferring a polynucleotide sequenceencoding an enzyme with carbohydrate degrading activity into E. colicells and subsequently rendering the cells competent using any of avariety of competency inducing procedures, including the standard highcompetency induction method described in Hanahan J. Mol. Bio.166:557-580 (1983) which comprises the step of washing the cells with abuffer comprising potassium acetate, MnCl₂, CaCl₂, glycerol, andhexamine cobalt chloride.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention described herein includes gram negative bacteria, such asE. coli cells that have been genetically modified so as to exhibitincreased transformation efficiency after being exposed to a competencyinducing procedure in comparison with similar E. coli cells lacking thegenetic construction. The cells of the subject invention have beenmodified by the addition of a genetic construction for the expression ofa carbohydrate degrading enzyme, and preferably a starch degradingenzyme such as alpha-amylase. The addition of the genetic constructionfor the expression of a carbohydrate degrading enzyme to E. coli cellsgives rise to E. coli cells that exhibit increased transformationefficiency after being rendered competent by a competency inducingprocedure. The invention also includes compositions of competent E. colicells containing a genetic construction for the expression ofcarbohydrate degrading enzymes, frozen compositions of such cells, andmethods for making the competent cells.

The term "starch-degrading enzyme" as used herein refers to enzymescapable of hydrolyzing at least one type of linkage present between theconstituent monosaccharide units of a carbohydrate molecule. The term"starch-degrading enzyme" as used herein refers to enzymes capable ofhydrolyzing at least one type of linkage present between the constituentmonosaccharide units of a starch molecule. The term "alpha-amylase" asused herein refers to enzymes capable of catalyzing hydrolysis of theα-1→4 glucosidic linkages of polysaccharides containing such glucosidiclinkages such as starch or glycogen. Preferred carbohydrate degradingenzymes are starch degrading enzymes. Preferred starch degrading enzymesare alpha-amylases. Particularly preferred starch degrading enzymes foruse in the invention are alpha-amylases isolated from thermophilicbacteria, especially the alpha-amylase gene from a recently isolateduncharaterized thermophilic bacterium. The polynucleotide sequenceencoding this alpha-amylase from the uncharacterized thermophilicbacterium can be found on the FAMY plasmid present in the E. colistrains having the ATCC accession numbers 69480, 69481, and 69482.

Genetic constructions containing a polynucleotide sequence encoding acarbohydrate degrading enzyme for use in the subject invention aredesigned so as to provide for the expression of a carbohydrate degradingenzyme. The expression of the carbohydrate degrading enzyme may beeither constitutive or inducible. Methods for expression of genes ofinterest in E. coli and other gram negative bacteria are well known. Forexamples of such techniques see Gene Expression Technology: Methods andEnzymology, Vol. 185, Goeddel, Editor, Academic Press, Incorporated, SanDiego, Calif. (1991). The genetic construction containing thepolynucleotide encoding the carbohydrate degrading enzyme of interestmay be designed to either replicate autonomously in a bacterial cell orbe incorporated into the genome of the bacterial cell. In addition topolynucleotide sequence encoding a carbohydrate degrading enzyme, thegenetic construction may comprise any one of a number of conventionalgenetic vectors such as plasmids, phages, phagemids, and the like. Thegenetic construction containing the polynucleotide encoding thecarbohydrate degrading enzyme may be introduced into an E. coli cell byany of a variety of transformation techniques including transformation,conjugation, electroporation, and the like.

Competent E. coli cells of the subject invention are produced bysubjecting E. coli cells containing a carbohydrate degrading enzymeencoding polynucleotide sequence for expression to a competency inducingprocedure. The term "competency inducing procedure" refers to anyprocedure used to render E. coli cells competent to transformation byexogenous DNA. The term "transformation efficiency" is a measure of thecompetence level of a given composition of competent cells.Transformation efficiency is expressed in terms of the number oftransformants obtained for each microgram (or other quantity) ofexogenous DNA added to a competent cell composition. It will beappreciated by the person of ordinary skill in the art that the measuredtransformation efficiency of a given composition of competent cellsusing a given transformation protocol will vary in accordance with theparticular exogenous DNA used in the transformation. Factors such as thesize and topology of the exogenous DNA can affect the measuredtransformation efficiency. Competency inducing procedures for E. coli(and other gram negative bacteria) are well known to the person ofaverage skill in the art of molecular biology. Although competencyinducing techniques vary considerably from one another, almost allcompetency inducing techniques involve the exposure of the cell tomultivalent cations and near 0° C. Competency inducing techniques foruse in the subject invention include, but are not limited to, the CaCl₂incubation method of Mandel and Higa (J. Mol. Bio., 53:159 (1970)), themethod of Hanahan, J. Mol. Bio., 166:557-580 (1983) that employs thestep of washing cells in a buffer comprising potassium acetate, KCl,MnCl₂, CaCl₂, glycerol, and hexamine cobalt chloride. The method ofHanahan is particularly preferred for use in the subject invention. Inaddition to Hanahan J. Mol. Bio. 166:557-580 (1983), a detailed protocolfor carrying out the standard high efficiency competency inductionmethod, which employs the step of washing cells in a buffer comprisingpotassium acetate, KCl, MnCl₂, CaCl₂, glycerol, and hexamine cobaltchloride, can be found in, among other places, U.S. Pat. No. 4,981,797(Jessee) and Sambrook et al., Molecular Cloning: a Laboratory Manual,2nd Edition, Cold Spring Harbor Press (1989). Competent cells of thesubject invention may be transformed using most well knowntransformation procedures, typically involving the step of exposingcompetent cells to a heat pulse in the presence of exogenous DNA.Examples of such transformation procedures can be found in Mandel andHiga, J. of Mol. Biol. 53:159 (1970) and Hanahan J. Mol. Bio.166:557-580 (1983).

After the E. coli cells have been rendered competent by a competencyinducing procedure, the cells may be frozen so as to retain theircompetence upon thawing. Frozen competent cells are a particularlyuseful embodiment of the invention because they may be stored forprolonged periods of time, thus avoiding the need to constantly producefresh preparations of competent cells. Protocols for preparing frozencompetent cells are known to the person of average skill in the art. Anexample of such a protocol can be found in Hanahan, J. Mol. Bio.166:557-580 (1983).

The introduction of a genetic construction for the expression of acarbohydrate degrading enzyme into E. coli serves to increase thetransformation efficiency of compositions of E. coli cells renderedcompetent for a wide variety of E. coli strains. The genotype of an E.coli cell strain containing a genetic construction for the expression ofa carbohydrate degrading enzyme may be selected so as to be particularlyuseful for a given genetic engineering experiment. For example, cloningvectors that are screenable because of LacZα fragment complementationmay contain a particular mutation within the LacZ gene. Similarly, thecell may contain various other deletions or mutations in order toprovide for complementation by the transforming DNA. The host cell mayeither possess or lack a restriction-modification system in order toexpedite cloning. The host cells may also lack one or more recombinationsystems, e.g., RecA, RecBC. Preferred E. coli strains for use in theinvention are cells that contain a mutation in the deoR gene, asdescribed in Hanahan, U.S. Pat. No. 4,851,348. Particularly preferredstrains of E. coli for use in the invention are the XL1-Blue™ strain(Stratagene, La Jolla, Calif.), the XL1-Blue MR strain, and the SURE™strain (Stratagene, La Jolla, Calif.) that have been modified by theaddition of a genetic construction for the expression of alpha-amylaseisolated from a thermophilic bacteria and have the ATCC accessionnumbers 69480, 69481 and 69482, respectively. The plasmid containing thealphaamylase gene in the E. coli strains having ATCC accession numbers69480, 69481 and 69482 may be readily transferred to other strains ofbacteria using techniques well known to the person of average skill inthe art. Similarly, the person of average skill in the art may excisethe alpha amylase gene from plasmids in the E. coli strains havingaccession numbers 69480, 69481 and 69482 and transfer the alpha amylasegene to a new genetic construct prior to transferring the gene to a newstrain of bacteria.

While reference is made to E. coli, other gram negative bacteria cellscan be rendered more competent, for example, bacteria from the GeneraPseudomonas, Rhizobium, Agrobacterium, Salmonella, Proteus, Shigella,Klebsiella and the like.

The invention having been described above, may be better understood byreference to the following examples. These examples are offered for thepurpose of illustrating the subject invention, and should not beinterpreted as limiting the invention.

EXAMPLES Example 1 Creation and Testing of Highly Competent E. ColiStrains Background

An alpha-amylase gene from a recently isolated uncharacterizedthermophilic bacterium was initially inserted onto an autonomouslyreplicating plasmid DNA element, and then was introduced into several E.coli strains through conjugation. This alpha-amylase gene can be foundin the plasmids present in the E. coli strains having the ATCC accessionnumbers 69480, 69481, and 69482. The resultant alpha-amylase genecontaining strains and the respective parent strain were renderedcompetent using the procedure of Hanahan (J. Mol. Biol. (1983)). Thetransformation efficiency of the alpha-amylase gene containing strainswere compared with the transformation of similar strains lacking thealpha-amylase gene.

Materials and Methods

The E. coli strains used in this work were the XL1-Blue, SURE™, XL1-MR,SCS-1, NM522, and BB4 strains. The genotype of these cells are asfollows: SURE™ e14⁻ (mcrA) , Δ (mcrCB-hsdSMR-mrr) 171, sbcC, recB, recJ,umuC:: Tn5 (Kan^(r)), uvrC, supE44, lac, gyrA96, relA1, thi-1, endA1 F'proAB, lacI^(Q) ZΔM15, Tn10, (tet^(r))!!; NM522 supE, thi-1, Δ(lac-proAB), Δ (hsdSMR - mcrB)5 (r_(K) -m_(k-)), F' proAB, lacI^(Q)ZΔM15!!; XL1-Blue recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1,lac, F'proAB, lacI^(Q) ZΔ M15, Tn10 (tet^(r))!!; XL1-Blue MRF' Δ (mcrA)183, Δ(mcrCB - hsdSMR - mrr) 173, endA1, supE44, thi-1, recAl , gyrA96,relA1, lac, F'proAB, laCI^(Q) Z Δ M15, Tn10 (tet^(r))!!; DH5α supE44, Δlac U169 (.0.80 lac Z Δ M15) hsdR17, recAl, endAl, gyrA96, thi-1,relAl!; SCS1: recΔ1, gyrA96, endA1, thi-1, hsdR17, supE44, relA1; BB4:e14⁻ (ΔmcrA), hsdR514, supE44, supF58, lacY1 or (lacIZY)⁶, galK2,galT22, metB1, trpR55, Δ (argF-lac)u169, F' lacI^(Q) ZΔM15proABTn10(tet^(r))!. The genotypes of these strains may also be found,among other places, in the appendix of the Stratagene (La Jolla, Calif.)1993 catalog.

Transformation and competency inducing procedures were essentially bythe standard high competency induction method described in Hanahan J.Mol. Bio. 166:557-580 (1983). Conjugal cell matings were carried out bymixing the donor and recipient cells of interest during theirexponential growth phase at an equimolar ratio, incubating at 37° C. for60 minutes with gentle agitation, and plating on the appropriateselection plates at 10⁻³ and 10⁻⁴ dilutions.

All DNA manipulations such as restriction enzyme digestion and ligationwere performed using enzymes obtained from Stratagene (La Jolla, Calif.)following the recommended conditions of the manufacturer.

Cloning alpha-amylase gene onto RSF1010 Derivative pAL205

pBM100, a pBluescript™ II vector derivative containing the amylase genefrom a thermophilic bacterium was digested with XbaI and HindIII and theappropriate DNA fragment was isolated and ligated to pAL205 digestedwith these same two enzymes. The ligation mix was transformed into SCS 1and Tet^(R) Cam^(R) colonies selected. Plasmid DNA from thesetransformants was isolated and subjected to restriction enzyme analysisto confirm the presence of the amylase gene.

Generation of FAMY by Homologous Recombination

E. coli strain BB4 (Rec⁺, NalS, Flacl^(q) Z M15, proAB, Tn10 (tetR), wastransformed with pAL205AMY DNA and Cam^(R) colonies were selected. BB4cells harboring pAL205AMY were then conjugally mated with XL1 MR cells(Nal^(R), F', Tet^(S), Cam^(S)) and Nal^(R), Tet^(R), Cam^(R)exconjugants selected. Since pAL205AMY is not capable ofself-transmissibility, the most likely means by which Cam^(R)exconjugants could be obtained was by a homologous recombination eventbetween the homologous Tet^(R) genes on both plasmids in the BB4 (Rec⁺)host prior to mating. The presence of the co-integrate plasmid wasconfirmed by mating this resultant strain, XL1-MRF AMY, with NM522 (F').All exconjugants from this "back" mating were simultaneously Tet^(R) andCam^(R). In addition, PCR amplification of the XL1 MRF AMY using amylasespecific primers yielded an amplification product that co-migrated on anagarose gel with a fragment amplified from pBM100 and pAL205AMY.

Transformation Protocol

The frozen competent cells produced by the method of Hanahan were thawedon ice. The cells were gently mixed by hand during thawing. 100 μlportions of the cells were placed into separated prechilled 15-ml Falcon2059 polypropylene tube. 1.7 μl of β-mercaptoethanol was added (at a1:10 dilution of stock 14.4M β-mercaptoethanol, diluted in high-qualitywater) to the 100 μl of bacteria, giving a final concentration of 25 mM.The contents of the tubes were swirled gently. The cells were thenincubated on ice for 10 minutes with gentle swirling every 2 minutes.Either 10 or 100 μg of supercoiled pUC18 DNA was added to the cells withgentle swirling. The tubes were then incubated on ice for 30 minutes.The tubes were then subjected to a heat pulse in a 42° C. water bath for30 seconds. The length of time of the heat pulse used here was importantfor maximizing transformation efficiency. The tubes were then incubatedon ice for 2 minutes. 0.9 ml of preheated (42° C.) SOC medium was thenadded and allowed to incubate at 37° C. for 1 hour with shaking at225-250 rpm. The transformation mixture was then transferred to platecontaining the appropriate medium and antibiotics spread onto thesurface of the plate.

MEDIA AND SOLUTIONS SOB Medium (per liter)

20.0 g of tryprone

5.0 g of yeast extract

0.5 g of NaCl

Autoclave

Add 10 ml of 1M MgCl₂ and 10 ml of 1M MgSO₄ /liter of SOB medium priorto use

Filter sterilize

SOC Medium (per liter)

SOB medium

Add 1 ml of a 2M filter-sterilized glucose solution or 2 ml of 20% (w/v)glucose prior to use

Filter sterilize

NZY Modified Medium (per liter)

5 g NaC1

6 g MgSO₄ ·7H₂ O

5 g yeast extract

10 g N·Z amine

Autoclave

LB Plate Solution (per liter)

10 g of tryptone

5 g of yeast extract

10 g of NaCl

15 g of agar

Autoclave

TY Medium (per liter)

8 g of tryptone

5 g of NaCL

5 g of yeast extract

Adjust to pH 7.2-7.4 with NaOH

Autoclave

YT Top Agar (per liter)

TY medium plus 6 g of agar

Autoclave

Cool to 55° C.

Add sterile MgSO₄ ·7H₂ O to a final concentration of 10 mM

TY Plates (per liter)

YT medium plus 15 g

Autoclave

Cool to 55° C.

Add sterile MgSO₄ 7H₂ O to a final concentration of 10 mM

LB-Ampicillin Plates (per liter)

Prepare the LB plate solution described above

Cool to 55° C.

Add ampicillin solution to a final concentration of 50 μg/ml (200 μg/ml)for TOPP 5 strain)

50 mg of ampicillin

10 ml of 1M MgSO₄

LB-Ampicillin-Methicillin Plate (per liter)

Prepare LB plate solution described above

Cool to 55° C.

Add 20 μg of ampicillin and 80 μg of methicillin

LB-Tetracycline Plates (per liter)

Prepare the LB plate solution described above

Cool to 55° C.

Add tetracycline solution

12-20 μg of tetracycline

10 ml of water

Store solution in a dark, cool place as this solution is light-sensitivewhile waiting of the LB plate solution to cool

Cover plates with foil if left out at room temperature for extended timeperiods to protect the plates from light

LB-Kanamycin Plates (per liter)

Prepare the LB plate solution described above

Cool to below 55° C.

Add the kanamycin solution

75 μg of kanamycin

10 ml of 1M MgSO₄

Store plates at 4° C.

RESULTS

Incorporation of the alpha-amylase gene into the genome of E. coli wasaccomplished by inserting the gene onto the F sex factor present inXL1-Blue, SURE, and XL1-Blue MRF strains. This method was superior toinserting the alpha-amylase gene onto the chromosome because thepresence of the alpha-amylase gene on the F episome enabled the conjugalmating of the gene into any E. coli strain of interest. Thealpha-amylase gene, initially cloned into pBluescript™ vector, wasisolated by restriction enzyme digestion and re-cloned onto an RSF1010plasmid (Bagdasarian et al, Gene 16:237-247 (1981)) derivative calledpAL205. Plasmid pAL205 is identical to plasmid pAL400 described inGreener et al. Genetics 130:27-36 (1992), except for the following 2modifications: (1) the luciferase gene has been replaced by thechloramphenicol resistance gene using HindIII and BglII restrictionendonucleases, and (2) a promoter element from Pseudomonas aeruginosahas been cloned into the BamHI cloning site. This plasmid harbors atetracycline resistance gene that is homologous to the tetracyclineresistance gene carried on the F episome residing in XL1-Blue and SUREstrains. Insertion of the amylase gene onto the F episome wasaccomplished in vivo, by homologous recombination between these twoplasmids in E. coli strain BB4. This new episome, termed FAMY, was thenconjugally mated into XL1-Blue, XL1-MR, and SURE™ strains to produce theultra competent cell derivatives.

Transformation efficiencies for XL1-MR FAMY, SURE FAMY, XL1-MR, SURE,and DH5 alpha (purchased from GIBCO/BRL) were determined by standardprocedures and are summarized in Table 1. The method of Hanahan (J. Mo.Bio. 166:557-580 (1983)) was used as the competency indices procedure.The data show that the FAMY derivatives of SL1-MRF and SURE had 4-5 foldgreater transformation efficiency than standard XL1-Blue, SURE and DH5alpha at equivalent DNA concentrations.

In order to determine that the other features present in the XL1-BlueFAMY and SURE FAMY were not perturbed, the cells were tested forinfectibility by M13 phage (indicating presence of F), and phage lambda.The FAMY derivatives were identical to the original parent. In addition,plasmid minipreps (both by standard alkaline lysis and by Stratagene'sClear Cut™ miniprep system) have been performed successfully theminiprep yields appear no different than the parental strains. Inaddition, the miniprep DNA was sequenced and the results were excellent.

The relative transformation efficiency of the FAMY derivatives withligated DNA as a substrate was tested. The results are shown in Table 2.

                  TABLE 1    ______________________________________    COMPETENT CELL TRANSFORMATION EFFICIENCIES    WITH SUPERCOILED DNA                             TRANSFORMANTS/    HOST      PICOGRAMS DNA  MICROGRAMS    ______________________________________    XL1-Blue  100            6.5 ± 0.3 × 10.sup.9    F' AMY    XL1-Blue  100            4.6 ± 0.6 × 10.sup.9    MRF' AMY    SURE F' AMY              100            3.9 ± 0.4 × 10.sup.9    XL1-Blue  100            1.2 × 10.sup.9    SURE      100              6 × 10.sup.8    DH5 alpha 100            8.0 × 10.sup.8    ______________________________________     TABLE 1:     Competent cells and transformation protocols were performed by the method     of Hanahan, J. Mol. Bio. 166: 557-580 (1983). 100 or 10 picograms of     supercoiled pUC18 DNA were added to 100 microliter aliquots of the cells     and plated onto LB plates containing 100 micrograms/ml ampicillin.

                  TABLE 2    ______________________________________    TRANSFORMATION EFFICIENCIES    WITH LIGATED DNA                       NUMBER OF    HOST               TRANSFORMANTS    ______________________________________    XL1-Blue         a     26                     b     33    XL1-Blue MRF' AMY                     a     78                     b     97    XL1-Blue F' AMY  a     130                     b     148    ______________________________________     TABLE 2:     Relative transformation efficiencies for XL1Blue and the derivatives. The     substrate DNA plasmid, 9703, and 8.78 kb M13 derivative, was digested wit     EcoRI restriction endonuclease, and incubated overnight with T4 ligase     prior to transformation. The A and B refer to the esults obtained in two     separate transformation experiments.

Example 2 Transformation of Pullanase Gene Containing Strains of E. Coli

A DNA segment isolated from the hyperthermophilic bacterium ES1 encodingthe starch-degrading enzyme pullalanase was cloned in to the Bluescriptplasmid vector (Stratagene, La Jolla, Calif.). This plasmid wastransformed into XL1-Blue cells (Stratagene, La Jolla, Calif.), whichwere then assayed by transformation with a chloramphenicol-resistantplasmid derivative of RSF1010 in order to determine whether expressionof pullalanase at 37° C. increased the transformation efficiency of thecells. Preliminary results obtained under non-optimal conditions for thepullanase gene were inconclusive in demonstrating an reproducibleimprovement in transformation efficiency. However, it is possible thatunder different growth conditions, e.g., higher than 37° C. (since thepullalanase was derived from a hyperthermophilic organism), thetransformation efficiency of the pullanase gene containing strain couldbe significantly higher than the control strain.

Biological Deposits

On Nov. 9, 1993, Applicants have deposited with the American TypeCulture Collection, Rockville, Md., USA (ATCC) strains XL1-Blue FAMY(ATCC accession #69480), XL1-BLue MR FAMY (ATCC accession #69481), andSURE™ FAMY (ATCC accession #69482). These deposits were made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the purposes of patent procedure andthe Regulations thereunder (Budapest Treaty). This assures maintenanceof a viable culture for 30 years from date of deposit. The organismswill be made available by ATCC under the terms of the Budapest Treaty,and subject to an agreement between Applicants and ATCC which assuresunrestricted availability upon issuance of the pertinent U.S. patent.Availability of the deposited strains is not to be construed as alicense to practice the invention in contravention of the rights grantedunder the authority of any government in accordance with its patentlaws.

Equivalents

All publications and patents mentioned in the above specification areherein incorporated by reference. The foregoing written specification isconsidered to be sufficient to enable one skilled in the art to practicethe invention. Indeed, various modifications of the above-describedmodes for carrying out the invention which are obvious to those skilledin the field of molecular biology or related fields are intended to bewithin the scope of the following claims.

What is claimed is:
 1. A method of preparing competent gram negativebacteria cells, said method comprising the steps of:transferring avector comprising a polynucleotide encoding the alpha-amylase genepresenton plasmid FAMY into gram negative bacteria cells, and treatingthe cells with a competency inducing procedure, whereby competent cellsare produced.
 2. The method according to claim 1, wherein the competencyinducing procedure is a standard high competency induction procedureemploying the step of washing the cells with a buffer comprisingpotassium acetate, KCl, MnCl₂, CaCl₂, glycerol, and hexamine cobaltchloride.
 3. The method of claim 1, said method further comprising thestep of freezing the competent cells.
 4. A competent cell produced bythe method of claim
 1. 5. A competent cell produced by the method ofclaim
 3. 6. A competent cell according to claim 4, wherein said gramnegative bacteria are E. coli.
 7. A competent cell according to claim 6,wherein the E. coli have a genotype selected from the group consistingof,(1) e14⁻ (mcrA), Δ (mcrCB-hsdSMR-mrr)171, SbcC, recB, recJ, umuC::Tn5 (Kan^(r)), Uvrc, supE44, lac, gyrA96, relA1, thi-1, endA1 F' proAB,lacI^(Q) ZΔM15, Tn10, (tet')!!, (2) recA1, endA1, gyrA96, thi-1, hsdR17,supE44, relA1, lac, F'proAB, lacI^(Q) ZΔ M15, Tn10 (tet^(r))!!, and (3)Δ (mcrA) 183, Δ(mcrCB - hsdSMR - mrr) 173, endA1, supE44, thi-1, recA,gyrA96, relA1, Lac, F'proAB, lacI^(Q) Z ≢ M15, Tn10 (tet^(r))!!.
 8. Acompetent cell according to claim 7, wherein said E. coli are selectedfrom the group consisting of ATCC accession #69480, ATCC accession#69481, and ATCC accession #69482.
 9. A competent cell according toclaim 5, wherein said gram negative bacteria are E. coli.
 10. Acompetent cell according to claim 9, wherein the E. coli have a genotypeselected from the group consisting of,(1) e14⁻ (mcrA), Δ(mcrCB-hsdSMR-mrr)171, SbcC, recB, recJ, umuC:: Tn5 (Kan^(r)), Uvrc,supE44, lac, gyrA96, relA1, thi-1, endA1 F' proAB, lacI^(Q) ZΔM15, Tn10,(tet')!!, (2) recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac,F'proAB, lacI^(Q) ZΔ M15, Tn10 (tet^(r))!!, and (3) Δ (mcrA) 183,Δ(mcrCB - hsdSMR - mrr) 173, endA1, supE44, thi-1, recA, gyrA96, relA1,Lac, F'proAB, lacI^(Q) Z ≢ M15, Tn10 (tet^(r))!!.
 11. A competent cellaccording to claim 10, wherein said E. coli are selected from the groupconsisting of ATCC accession #69480, ATCC accession #69481, and ATCCaccession #69482.